The chemical biogeography of a widespread aromatic plant species shows both spatial and temporal variation

Abstract Plants produce a wide variety of secondary metabolites, but intraspecific variation in space and time can alter the ecological interactions these compounds mediate. The aim of this work was to document the spatial and temporal chemical biogeography of Monarda fistulosa. I collected leaves from 1587 M. fistulosa individuals from 86 populations from Colorado to Manitoba, extracted and analyzed their terpenes with gas chromatography, mapped monoterpene chemotypes, and analyzed chemical variation with principal component analysis. I also measured the amounts of terpenes in different plant tissues to examine intraplant variation and monitored leaf terpene chemistry over a single growing season to examine temporal patterns. Finally, I extracted terpenes from herbarium samples up to 125 years old and compared the chemotypes with recent samples from the same sites. M. fistulosa populations consisted mostly of thymol (T) and carvacrol (C) chemotypes, but geraniol (G) and (R)‐(−)‐linalool (L), a chemotype new to this species, were also present. A principal component analysis showed that most of the chemical variation across populations was due to the amounts of the dominant terpene in plants. Intraplant tissue chemistry revealed that leaves mostly had the greatest amounts of terpenes, followed by floral structures, stems, and roots. Short‐term temporal variation in leaf chemistry of T and C plants over a growing season showed that plants produced the highest levels of biosynthetic precursors early in the season and their dominant monoterpenes peaked in mid‐summer. Plant chemotype was discernable in the oldest herbarium samples, and 15 of 18 historic samples matched the majority chemotype currently at the site, indicating that population chemotype ratios may remain stable over longer time scales. Overall, the results show that plant species' secondary chemistry can vary both spatially and temporally, which may alter the biological interactions that these compounds mediate over space and time.


| INTRODUC TI ON
Plants are heterogeneous resources for the organisms with which they interact and one major source of plant heterogeneity is variation in the amounts and composition of the secondary metabolites that they produce. As the name implies, secondary metabolites are not part of primary plant metabolism (e.g., sugars, proteins, nucleic acids) but are specialized compounds (e.g., alkaloids, terpenoids), often with high taxonomic affinity. Variation in plant secondary chemistry can occur at many scales, both spatially and temporally. Spatial variation in secondary metabolites can occur at the scale of a single individual with differences among different plant tissues (Bowers & Stamp, 1992), among individuals within populations (Keefover-Ring et al., 2009), and among different populations, up to regional and large geographic scales Pratt et al., 2014). Temporal variation can happen over rapid time scales, such as induction (Karban & Baldwin, 1997;Keefover-Ring et al., 2016), with plant ontogeny (Barton & Koricheva, 2010), or over longer evolutionary time scales, as the selective forces acting on plant populations change over time (Thompson et al., 2013).
From an ecological point of view, understanding the spatial and temporal variation of the secondary chemistry of a species is important since these compounds are involved in many biological interactions between the plants that make them and other species.
The interactions that these chemicals mediate can be either mutually beneficial, such as the attraction of pollinators, or antagonistic, such as the deterrence of herbivores and allelopathy (Gershenzon & Dudareva, 2007;Langenheim, 1994). Furthermore, these interactions often operate bi-directionally, such that the organisms surrounding a plant can also affect the chemistry of the plant population (Linhart, 1991). In addition to biotic forces, a plant's secondary chemical composition can also be shaped by abiotic forces, such as nutrients (Loney et al., 2006), moisture availability (Johnson et al., 1997), or temperature (Pratt et al., 2014;Thompson et al., 2007), and these forces can vary over the landscape and with time, resulting in different secondary chemistry-driven ecological and evolutionary outcomes (Thompson, 2005). However, large-scale studies to determine the phytochemical landscape (Hunter, 2016) of a species can be time-consuming and costly and have only been done for a limited number of species (e.g., Bohm, 2009;Gouyon et al., 1986;Keefover-Ring et al., 2014).
The focus of this work is to document the spatial and temporal chemical biogeography of one plant species, Monarda fistulosa L. var. menthifolia, in at least part of its extensive range. Like many species in the Lamiaceae, M. fistulosa synthesizes essential oils (a mixture of mono-and sesquiterpenes) in mostly peltate granular trichomes on the surfaces of flower petals, calyces, bracts, leaves, and even stems (Heinrich, 1973;Pfab et al., 1980). The pattern of essential oil production in M. fistulosa is a chemical polymorphism where individuals are identified with distinct chemical phenotypes, or chemotypes, where a single monoterpene dominates (Keefover-Ring et al., 2009), which is consistent with other labiate species (Fleisher & Sneer, 1982;Vernet et al., 1986). Except for the work of Marshall and Scora (1972) and Keefover-Ring (2015), however, all other studies documenting M. fistulosa chemistry analyzed relatively few individuals.
The largest spatial scale in this study includes mapping the chemotypes of M. fistulosa populations from southern Colorado and extending north to sites in Wyoming, the Dakotas, and Manitoba, which included collecting and analyzing 1587 individuals from 86 separate populations. On a much smaller spatial scale, the amounts of terpenes in different plant tissues were measured to examine in- The specific questions addressed in this study were: (1) (Prather et al., 2002), commonly known as wild bergamot, bee balm, or horse mint, is a perennial mint that occurs in all of the continental United | 3 of 19 KEEFOVER-RING that are aliphatic in character, thymol and carvacrol are based on a phenolic structure and are only produced by a few other labiate species, including species in the genera of Thymus and Oregano (Chizzola, 2003;Fleisher & Sneer, 1982;Skoula & Grayer, 2005;Stahl-Biskup & Saez, 2002), and other species in the genus Monarda (Burt, 1936;Scora, 1967). Plants that produce thymol and carvacrol almost always also have relatively high amounts of the aliphatic monoterpenes γ-terpinene and p-cymene. γ-Terpinene serves as the biosynthetic precursor for thymol and carvacrol and p-cymene results from dehydration of a dienol intermediate that leads to carvacrol (Krause et al., 2021; Figure 1).

| Sample collection and preparation
To investigate geographic variation in M. fistulosa essential oil chemistry, I collected single leaves from 1587 individual plants at 86 different populations for terpenoid analysis from 2002 to 2007 (Table A1).
The majority of the populations sampled were located in Colorado (66 sites) and specifically in Boulder County (41 sites). There were also 14 sites in southern Manitoba, Canada, three in North Dakota, two in South Dakota, and one in Wyoming. The number of individuals per site ranged from one to 122 with a mean of 18 (SD = 14.2).
Sites were chosen using collection records from the University of Colorado Museum Herbarium (COLO) and by observing populations, usually in flowers, when driving or hiking. At each site, plants were haphazardly chosen with enough distance between them to ensure that the stems sampled were from different individuals. Typically, the first leaf immediately below the bracts was detached next to the stem, rolled to fit into a 2 ml microcentrifuge tube, and completely submerged with either 1.00 or 1.50 ml of ethanol, containing m-xylene as an internal standard (0.1 μl/ml). Usually, the entire procedure was carried out in the field within a few hours of collection, or when not possible within 24 h of collection, during which time leaf samples were stored in either a small cooler with ice or a refrigerator. Upon return to the lab, samples were placed in a sonication bath for 15 min and then allowed to extract for 7 days at room temperature. After the 7-day soaking period, 100 μl of the solution from each sample was combined with 100 μl of internal standard solution and injected into a gas chromatograph (GC). Leaves were later removed from the solvent, dried to a constant weight at 70°C, and weighed to the nearest mg.

| Statistical analysis
All statistical analyses in this study were carried out using SAS ver- samples in the chemotype mapping part of the study using dominant compound peak areas, however, the leaf dry weights needed to calculate compound concentrations were only available for 1007 samples. Thirty-nine of these sites were from Colorado (including the L chemotype site), 14 from Manitoba (including all sites containing G chemotype plants), three from North Dakota, two from South Dakota, and one from Wyoming.

| Sample collection and preparation
To assess the essential oil chemistry of various tissues (intraplant) of M. fistulosa, single stems from 13 separate C chemotype plants, including some roots, were collected from the Crescent Meadows (Table A1) population in Colorado on July 18, 2003. The stems were dissected and various parts were soaked separately in 1.00 ml internal standard solution in 2 ml microcentrifuge tubes. Separate parts included flowers (including petals and all sexual parts), calyces, the bracts that subtend the inflorescence, leaves from the first, second, and third positions below the bracts, stems, and roots. This process was repeated on July 28, 2004, with single stems from five separate geraniol chemotype plants from Spruce Woods Provincial Park in Manitoba. This time the parts used included only flowers, calyces, bracts, only the second leaf below the bract, and stems. All intraplant samples were analyzed by GC, as above.

| Statistical analysis
The proc GLM function was used to test for differences in γterpinene, p-cymene, thymol, carvacrol, and total terpenes between the various parts of the 13 C chemotype plants. Compounds with significant ANOVAs were then subjected to pair-wise comparisons with a Ryan-Einot-Gabriel-Welsch multiple range test. Since carvacrol methyl ether is usually either present or absent in individuals, only the three plants in which it occurred were used in the statistical analysis of this compound. The same statistical procedure was used to look for chemical differences among parts of the five G chemotype plants. In this case, only the main monoterpene geraniol and total terpenes were examined. To meet ANOVA assumptions of normality individual terpene data were square root transformed.

| Sample collection and preparation
To determine the changes in levels of terpenes that occur over an

| Statistical analysis
I performed individual repeated measures analyses for γ-terpinene, p-cymene, thymol, carvacrol, and total terpenes separately for the two different chemotypes for the five different time periods using PROC MIXED. Compounds that were significant for the repeated factor of time were then subjected to pair-wise comparisons with a Ryan-Einot-Gabriel-Welsch multiple range test. To meet ANOVA assumptions of normality individual terpene data were transformed as above. varied among the accessions, with generally more specific location placement for the more recent samples. However, even some of the earliest sites sampled were well-described.

| Sample collection and preparation
Samples for chemical analysis consisted of approximately onehalf of a leaf (usually the first leaf below the bracts) cut from each pressed specimen and placed in separate small manila envelopes.
Eight to twelve milligrams of the dried leaf material was weighed to 0.1 mg, placed in small glass vials with PTFE-lined screw tops, and 0.50 ml of an internal standard solution (m-xylene in n-hexane, GC 2 hexane, Burdick and Jackson) was added and samples sonicated for 15 min. Samples were allowed to soak for 7 days before analysis.
Twenty of the historic sites were also locations where contemporary plants were collected for the chemical biogeography work described above (Table A2). Many of the remaining historic sites were revisited, but either the exact location of the population could not be found or no current M. fistulosa population still existed.

| Chemical analysis
Historic samples were analyzed with an Agilent 6890 N GC/Agilent 5975 MS with an HP-1MS column (30 m × 0.25 mm I.D., film thickness 0.25 μm, Agilent Technologies, Inc.). One microlitre of each sample was injected in the splitless mode with oven conditions that included an isothermal hold at 60°C for 5 min, followed by a ramp of 10°C/min to 250°C. Linear retention indices were also calculated on the HP-1 column with the same oven conditions used when determining them on the DB-Wax column.

| Statistical analysis
I used a χ 2 statistic to test whether the observed chemotypes from herbarium samples differed from the chemotypes expected based on the majority of chemotype detected at the sites from the recent sampling. Also, while many of the historical samples contained very small amounts of several different terpenes, only p-cymene, thymoquinone, carvacrol methyl ether, thymol, and carvacrol were present in relatively large amounts in most of the samples and were the only plant terpenes used for statistical analyses. Using the PROC REG function, the concentration data for p-cymene, thymoquinone, carvacrol methyl ether, a total of the two phenolic monoterpenes (thymol and carvacrol), and a total of all five were regressed on the sample collection date.

| Colorado
The majority of the populations sampled were located in Colorado  (Table 1). Plants with increasing amounts of thymol (concentration, not percentage) corresponded to increasingly negative PC 1 values. C chemotype individuals showed a similar relationship, but higher positive PC 1 values were correlated with greater carvacrol amounts. In addition to variation in thymol and carvacrol amounts, PC 2 also included the variation in geraniol concentrations of the G chemotype plants from Manitoba. The three L plants were also a small part of the variation of PC 2. G and L plants both had higher amounts of their main monoterpene than T and C plants and a much higher percentage of the total essential oil (Table 1)

| Intraplant chemical variation
Different M. fistulosa plant tissues varied in their content and composition of terpenes (Figure 4). In most cases, stems and roots differed from all other parts and had the lowest concentration of essential oils. In the 13 C chemotype plants analyzed, γ-terpinene differed between the various tissues (F 7,96 = 59.84, p < .001) and was found at its highest levels in flowers, calyces, and leaves, all of which differed from bracts, stems, and roots ( Figure 4a). The lowest amount of this compound was in stems and roots, which differed from bracts.

| Comparing chemotypes of historic and contemporary sites
All of the historic M. fistulosa herbarium samples contained measurable amounts of terpenes and plant chemotype was able to be deter- in Greece revealed that although the total amount of essential oil in plants of both T and C chemotypes could be partially explained by elevation, summer water deficiency, and thermal efficiency; the spatial distribution was not correlated with any of these factors (Vokou et al., 1993). By contrast, in reciprocal transplant experiments of Thymus vulgaris in southern France, Thompson et al. (2007) found that not only are T and C plants restricted to warmer areas  (Hahn et al., 2021). Also, while T and C chemotypes seem to be more resistant overall to herbivores than other chemotypes (Linhart & Thompson, 1999)  . T. vulgaris also contains the same four chemotypes that I found in M. fistulosa with the order of dominance determined to be: G > L > C > T. This pattern also seems to be the case in the genus Monarda, at least for G, L, and T individuals (Marshall & Scora, 1972).

| Temporal variation of M. fistulosa chemistry
The short-term temporal changes in M. fistulosa chemistry seen dur- notable differences among chemotypes were seen in samples collected on June 1 that showed T plants had significantly higher amounts of total terpenes, due mostly to a much higher amount of the phenolic precursor γ-terpinene. While the chemical defense of labiates is usually thought to be constitutive, these results show that the chemical phenotype of these plants is quite variable during a single season and this may present a "moving target" to potential herbivores (Adler & Karban, 1994), or even to pollinators, since the changing amounts of the more volatile monoterpenes (γ-terpinene and p-cymene) in plant tissues during the season will lead to temporal changes in terpene emissions (Keefover-Ring, 2013). Finally, the seasonal change in essential oil composition also indicates that the allelopathic potential of M. fistulosa may change over the growing season (Linhart et al., 2015). While material from herbarium samples is routinely used for molecular genetic analysis, this resource has less often been used to assess the past chemical diversity of a species (Almasirad et al., 2007;Baser et al., 2005;Novak et al., 2002), and only rarely in an ecological context (Berenbaum & Zangerl, 1998;Zangerl & Berenbaum, 2005). (2005)  However, herbarium samples will lose even these less volatile terpenes (thymol and carvacrol) over time, as can be seen from the regressions of compound content versus sample collection year. In addition, differential loss of terpenes due to very dissimilar volatilities (Keefover-Ring, 2013), combined with compound degradation, such as the conversion of thymol and carvacrol to thymoquinone (Jukic & Milos, 2005;Krause et al., 2021), led to results that cannot be directly compared with the exact chemical profiles of contemporary plants. Thus, due to the physical properties of mono-and sesquiterpenes, herbarium samples containing essential oils can probably only be used in qualitative comparisons to contemporary material (Novak et al., 2002).

Zangrel and Berenbaum
The appearance of a new trait in a population may lead to evolution in that species if that trait possesses some advantage over an existing one. In the case of essential oil phenotypes in M. fistulosa, for a large extent of its range, the T and C chemotypes have been very successful in remaining the only plant chemotypes, possibly due to the greater toxicity these compounds have shown toward a range of herbivores and parasites , 1999, and plant competitors (Linhart et al., 2015;Tarayre et al., 1995), compared with other monoterpenes. However, two other chemotypes of M. fistulosa have arisen-G chemotype plants in Canada and L chemotype plants in Colorado. Marshall and Scora (1972) were the first to formally document the existence of a geraniol chemotype of wild bergamot in and around Spruce Woods Provincial Park, known to exist since at least the mid-1950s. They theorized that the G chemotype probably represents a recent mutation in the species, since it is very restricted in its distribution, but can still readily interbreed with other M. fistulosa chemotypes (Marshall & Scora, 1972). likely that these plants may also represent a very recent mutation (Marshall & Scora, 1972). Unlike the new chemical race of geraniol plants in Canada, the fate of this new chemotype remains to be seen, although they were still at the site in the summer of 2019 (Keefover-Ring, personal observation). The process of evolution is a series of trials with new phenotypes, and sometimes these experiments fail. While evidence from other labiates shows that T and C chemotypes certainly have an advantage over nonphenolics with respect to withstanding herbivory (Linhart & Thompson, 1999) or inhibiting competitors (Linhart et al., 2015), G and L chemotypes may possess characteristics that may favor them. For instance, both of these chemotypes produce monoterpenes (linalool and geraniol) known to attract pollinators (Andersson & Dobson, 2003;Schmidt, 1999) or deter herbivores, such as grasshoppers (Linhart & Thompson, 1999) or aphids (Linhart et al., 2005). Also, data from Marshall and Scora (1972) hinted that the inheritance pattern of M. fistulosa chemotypes may be G > L > C > T, as is the case in T. vulgaris . If so, it is likely that the new chemotypes will continue to expand their range since any pollen transfer from G and L plants to C or T plants will mostly result in G or L offspring (Marshall & Scora, 1972;Vernet et al., 1986). Recent evidence shows that M. fistulosa individuals with chemotypes other than T or C may be more widespread than previously thought. In the summers of 2020 and 2021, I detected a few G and L plants in remnant or restored prairie sites near Madison, WI (Keefover-Ring, personal observation). In addition, at one of these sites, several plants were found containing 1,8-cineole (eucalyptol; scent of eucalyptus) and α-terpineol (pine-like scent), which alternated as the largest peak in their essential oil profile (Keefover-Ring, personal observation).
While these chemotypes have never been described in M. fistulosa, Scora (1967) found relatively high levels of both of these monoter-

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
PCA site comparison and chemistry data: Dryad DOI https://doi. org/10.5061/dryad.18931 zd12.

A PPEN D I X A
The chemical biogeography of a widespread aromatic plant species shows both spatial and temporal variation